NUCLEIC ACID PROBES FOR LACTOBACILLUS DETECTION

Abstract: Nucleic acid sequences which preferentially bind to the rRNA or rDNA of microorganisms which cause the spoilage of beer are disclosed. The beer spoilage microorganisms are predominantly of the genera Lactobacillus and Pedicoccus. The nucleic acids may be used as probes in assays to detect the presence of these microorganisms. Kits containing two or more probes are also described.

Description: NUCLEIC ACID PROBES FOR LACTOBACILLUS DETECTION This invention relates to nucleic acids, probes, kits, and methods for the detection of organisms, including Pediococcus sp. and Lactobacillus sp. which are involved with the spoilage of beer in the brewing environment. Background of the Invention The prevention of beer-spoilage by contaminating microorganisms is a major concern of commercial breweries. The predominant organisms which have been shown to spoil beer, or which have been associated with beer-spoilage are members of the genera Lactobacillus and Pediococcus (see The Prokaryotes. Vol. II. 2nd Edition, Balows, et al, Eds., 1991). These bacteria may be present in very low numbers and their detection may require three to five days or more by traditional culture methods. Members of the genus Pediococcus are Gram-positive cocci which frequently form tetrads. They have complex nutritional requirements and are capable of fermenting a variety of sugars. They are facultative anaerobes found in a variety of habitats, most frequently associated with fermenting vegetation. There are eight species in this genus; P. damnosus is the primary member of the genus known to cause beer spoilage. The genus Lactobacillus contains Gram-positive nonsporulating rods, utilizing strictly fermentative metabolism and having complex nutritional requirements. They are found in a variety of habitats, including water, dairy, meat and fish products, vegetation and fermenting vegetation, and in the mouth and intestinal tract of mammals. Several studies have identified bacterial strains capable of spoiling beer, and the relative numbers of strains within the species so implicated were, in decreasing order of importance: Lactobacillus brevis, P. damnosus, L. casei, L. lindneri, L. corynifonnis, L. buchneή, L. plantarum, and L. curvatus. The current methods of detection of beer-spoilage organisms rely on classical microbiology and a general determination of the presence or absence of contamination by bacteria. These methods include: (a) culture, (b) direct fluorescence antibody (DFA), and (c) nucleic acid probes for culture confirmation. Actual identification of spoilage organisms requires classical biochemical tests and fulfillment of Koch's postulates, i.e. "reinfecting" fresh beer and showing it to become spoiled. Description of the Invention One aspect of this invention is to provide nucleic acids complementary to unique nucleic acid sequences within the ribosomal RNA (rRNA) and DNA (rDNA) of organisms which cause beer spoilage, but are not present in unspoiled beer. It is another aspect of this invention to provide nucleic acid probes which can hybridize to target regions which can be rendered accessible to probes under normal assay conditions. It is a further aspect of the invention to provide for probes which either (1) specifically discriminate between P. damnosus and non-Pediococcus species; (2) specifically discriminate between the majority of Pediococcus strains causing beer-spoilage and other species; (3) specifically discriminate between L. brevis and non-Lactobacillus species; (4) specifically discriminate between a cluster of Lactobacillus species (the cluster being a group of bacteria consisting of L. frucύvorans, L. casei, L. curvatus, L. brevis, and L. buchneή) and non-cluster species; (5) specifically discriminate between the group of P. damnosus and L. brevis and other species; (6) specifically discriminate between the majority of Pediococcus and Lactobacillus species causing beer spoilage and other species; or (7) specifically discriminate between the majority of Pediococcus and Lactobacillus (and related species) and other species. Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli are referred to as 5S, 16S, and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S and 5.8S. These names are historically related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, rRNA molecules vary substantially in size between organisms. This notwithstanding, 5S, 16S and 23S rRNA are art-recognized names referring to rRNA molecules in any bacteria and this convention will be used herein. Description of the Figures Figure 1 is a diagram of a sandwich assay. Definitions As used throughout the application and claims, the term "probe" will refer to synthetic or biologically produced nucleic acids, of 10 to 250 bases in length, which by design or selection, contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequen¬ ces, and optionally contain a moiety for detection or enhancing assay performance. A minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and form stable hybridization products, and a maximum of 250 nucleotides generally represents an upper limit of nucleotides in which reaction parameters can be adjusted to determine mismatched sequences and preferential hybridization. Therefore, in general, a preferred length of a probe will be between 10 and 250 nucleotides. Probes may also optionally contain certain constituents that pertain to their proper or optimal functioning under certain assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (such as by end-capping), to carry detection ligands (such as fluorescein, 32 P, biotin, etc.) or to facilitate their capture onto a solid support (e.g. poly-deoxyadenosine "tails"). "Preferential hybridization" or "hybridizing preferentially" is to be used in a relative sense; i.e. one hybridization reaction product is more stable than another one under identical conditions. Under some conditions, a hybridization reaction product may be formed with respect to one target, but not another potential binding partner. It is well within the skill of the ordinary artisan to compare stability of hybridization reaction products and evaluate which one is more stable, i.e. determine which one has bound "preferentially". As used herein, the terms "homology" and "homologous to" are meant to refer to the degree of similarity between two or more nucleic acid sequences, and is not meant to imply any taxonomic relatedness between organisms. The degree of similarity is expressed as a percentage, i.e. 90% homology between two sequences will mean that 90% of the bases of the first sequence are identically matched to the bases of the second sequence. A "cluster of Lactobacillus species" means a group of Lactobacillus species selected from the group consisting of L. fi ctivorans, L. casei, L. curvatus, L. brevis, and L. buchneή. "Specific" means that a nucleotide sequence will hybridize to a defined target sequence and will substantially not hybridize to a non-target sequence, or that hybridiza¬ tion to a non-target sequence will be minimal. "Hybridization" is a process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another. "Substantial hybridization" means that the amount of hybridization will be to an extent that one observing the results would consider the result positive in a clinical setting. Data which is considered "background noise" is not substantial hybridization. "Stringent hybridization conditions" mean approximately 35 ° C to 65° C in a salt solution of approximately 0.9 molar NaCl. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which a hybridization is to take place will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. "Lactobacillus sp." refers to any member of the genus Lactobacillus, regardless of the species. "Pediococcus sp." refers to any member of the genus Pediococcus, regardless of the species. "Majority of when referring to strains means more than half of the strains known or, more than half of the strains tested, when one tests a representative sampling of at least 25 strains. When referring to species, it means more than one half of the known species, or more than one half of the species tested, when one tests a representative number of species.. In accordance with this invention, there are provided nucleic acids having approximately 10 to 250 nucleotides which (1) hybridize preferentially to rRNA or rDNA of P. damnosus as compared to other non-Pediococcus species; (2) hybridize preferen¬ tially with the majority of Pediococcus strains causing beer-spoilage compared to other species; (3) hybridize preferentially with L. brevis compared to -.on-Lactobacillus species; (4) hybridize preferentially with a cluster of Lactobacillus species (selected from the group consisting of: L. fructivorans, L. casei, L. curvatus, L. brevis, and L. buchneή) com¬ pared to other species; (5) hybridize preferentially with the group of P. damnosus and L. brevis compared to other species; (6) hybridize preferentially with the majority of Pedio¬ coccus and Lactobacillus species causing beer spoilage as compared to other species; and (7) specifically discriminate between the majority of Pediococcus and Lactobacillus (and related species) and other species. Under those same hybridization conditions, the nucleic acids of this invention do not substantially hybridize to the rRNA or rDNA of non-target organisms, or the host or environmental matrix which may be present in test samples. The nucleic acids of this invention are useful for detecting the presence of an organism which would cause spoilage in beer. Probes which are either complementary to or at least 90% homologous to at least ten consecutive nucleic acids of the aforemen¬ tioned nucleotides also form another aspect of this invention. One embodiment of this invention are nucleic acids and probes which are homologous to or hybridize to regions of 16S rRNA or rDNA of beer-spoiling micro¬ organisms. The regions of 16S rRNA of particular interest are in reference to the numbering of the homologous regions in E. coli, a standard well known to those of ordinary skill in the art, include: P. damnosus: 16S rRNA positions 285 to 320, 450 to 485, and 1435 to 1470; L. brevis: 16S rRNA positions 75 to 105, and 450 to 485; P. damnosus or L. brevis: 16S rRNA position 805 to 840; Pediococcus and Lactobacillus: 16S rRNA positions 120 to 150, 210 to 245, 280 to 315, 485 to 515, and 750 to 785. Another embodiment of this invention is nucleic acids and probes which hybridize to regions of 23S rRNA or rDNA of beer-spoiling microorganisms. The regions of 23S rRNA of particular interest are in reference to the numbering of the homologous regions in E. coli, a standard well known to those of ordinary skill in the art, include: P. damnosus 23S rRNA positions 700 to 740, 870 to 910, 925 to 960, 1130 to 1165, and 1205 to 1245. L brevis 23S rRNA positions 280 to 320, 325 to 363, 1130 to 1165, 1265 to 1300 and 1480 to 1512. P. damnosus and L. brevis 23S rRNA positions 600 to 635. Preferably the nucleic acid composition is complementary to or homologous with at least 90% of a sequence comprising any ten consecutive nucleotides within sequences selected from the group of sequences defined by the group of probes consisting of: 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891, 2892, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. The sequences of these probes are presented below. A further embodiment of this invention includes a kit for the detection of the presence of beer-spoiling microorganisms. The kit comprises a set of nucleic acids comprising at least two nucleic acids. Each nucleic acid is of 10 to 250 nucleotides and is of a different base sequence composition. Each nucleic acid is complementary to or homologous with at least 90% of a sequence comprising any ten consecutive nucleotides selected from the group of sequences defined by probes 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891, 2892, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. A set of nucleic acids is particularly suited for detecting beer-spoiling microorganisms in a two probe, sandwich assay. The kit additionally comprises reagents, compositions, instructions, disposable hardware and suitable packaging to allow market¬ ing in a convenient assembly. A further embodiment of the present invention includes methods for the detection of the presence of beer-spoiling microorganisms. The method comprises the steps of contacting a sample suspected of containing a target with at least one nucleic acid. The nucleic acid has approximately 10 to 250 nucleotides which hybridize preferentially to rRNA or rDNA of: (1) P. damnosus; (2) the majority of Pediococcus strains causing beer-spoilage, but not other species; (3) L. brevis, but not other Lactobacillus species; (4) a cluster of Lactobacillus species (comprised of L. fructivorans, L. casei, L. curvatus, L. brevis, and L. buchneή), but not other species; (5) the group of P. damnosus and L. brevis, but not other species; (6) the majority of Pediococcus strains and Lactobacillus species which cause beer spoilage, but not other species; and (7) the majority of Pedio¬ coccus and Lactobacillus (and related species) but not other species. The method includes the steps of imposing hybridization conditions on the sample such that the nucleic acid binds preferentially to the target rRNA or rDNA to form nucleic acid complexes and detecting the complexes as an indication of the presence of the target organism(s). Preferably, the nucleic acid of the present invention is at least 90% homologous to a sequence comprising any ten consecutive nucleotides selected from the group consisting of sequences defined by probes 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891,2892, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. The probes of the present invention provide the basis for development of a nucleic acid hybridization assay for the specific detection of beer-spoilage organisms, in beer or in environmental samples. The probes of the present invention also form the basis for confirmation of the presence of microorganisms which have been shown to spoil beer. The first step taken in the development of the probes of the present invention involved the identification of the regions of 16S or 23S rRNA which potentially could serve as target sites for specific nucleic acid probes with the desired sensitivity. This included discovering which probe target sites were unique to: 1) P. damnosus; 2) the majority of Pediococcus strains causing beer-spoilage; 3) L. brevis; 4) a subgroup of the Lactobacillus sp.; 5) the group of P. damnosus and L. brevis; 6) the group of the majority of Pediococcus and Lactobacillus species which have been shown to spoil beer; and 7) the group of the majority of Pediococcus and Lactobacillus and related species. This involved finding sites which are: 1. different between P. damnosus and other Pediococcus and noτi-Pediococcus species; 2. different between the majority of Pediococcus strains tested and other species; 3. different between L. brevis and other Lactobacillus and non-Lactobacillus species; 4. different between a cluster of Lactobacillus species ( . fructivorans, L. casei, L. curvatus, L. brevis, and L. buchneή) and other species; 5. different between the group of P. damnosus and L. brevis and other species; 6. similar for all organisms which have been shown to cause beer-spoilage as demonstrated by a representative sampling of 25 strains, but different between the next closest evolutionary neighbors' sequences; and 7. similar between the majority of Pediococcus and Lactobacillus and related species, but different from other species except for L. minutus, L. lacti, members of the Micrococcus genus and members of the Pectinatus genus. To accomplish the above analysis, precise alignments of P. damnosus and L. brevis 16S and 23S rRNA sequences were developed. The essentially complete 16S and 23S rRNA sequences of both P. damnosus and L. brevis were determined using standard laboratory protocols. The rDNAs so obtained were cloned into plasmid vectors from products produced by enzymatic amplification (such as that described in Weisburg, 1991, J. Bacteriol. 173:697-703, which is incorporated herein by reference). T e P. damnosus and L. brevis sequences were aligned with homologous sequences of other Lactobacillus species, Gram-positive organisms and other eubacterial rRNA sequences including E. coli (which are widely used as standard reference sequences by those of ordinary skill in the art). Based on the determined 16S and 23S rRNA sequences of P. damnosus and L. brevis, twenty-two probes were designed, synthesized, and tested. The specific behaviors of the probes are dependent to a significant extent on the assay format in which they are employed. Conversely, the assay format will dictate certain of the optimal features of the particular probes. The discovery that probes could be generated with the extraordinary inclusivity and exclusivity characteristics of the present invention with the respect to P. damnosus and L. brevis without incurring undesirable levels of cross-reactivity was unpredictable and unexpected. The first group of preferred probes are able to differentiate between P. damnosus and other species. P. damnosus Specific 16S rRNA Probes P. damnosus Probe 2858 (28mer, 46% G+C) (SEQ ID NO:l) 5'-TCA CAG CCT TGG TGA GCC TTT ATC TCA T-3' P. damnosus Probe 2861 (29mer, 48% G+C) (SEQ ID NO:2) 5'-CAC TGC ATG AGC AGT TAC TCT CAC ACA CT-3' P. damnosus Probe 2867 (28mer, 61% G+C) (SEQ ID NO:3) 5'-CGG CTA GCT CCC GAA GGT TAC TCC ACC T-3' A second group of preferred probes are able to detect the majority of Pediococ¬ cus beer-spoilage strains. Majority of Pediococcus Genus 23S rRNA Probes Pediococcus Genus Probe 2876 (32mer, 50% G+C) (SEQ ID NO:4) 5'-CCA CAG TCT CGG TAA TAT GTT TAA GCC CCG GT-3' Pediococcus Genus Probe 2877 (31mer, 58% G+C) (SEQ ID NO:5) 5'CGC TCC AAC AGT CCT CAC GGT CTG CCT TCA T-3' A third group of preferred probes are specific for L. brevis. L. brevis Specific 16S rRNA Probes L. brevis Probe 2868 (28mer, 43% G+C) (SEQ ID NO:6) 5'-CAA CGT CTG AAC AGT TAC TCT CAA ACG T-3' L. brevis Probe 2869 (32mer, 41% G+C) (SEQ ID NO:7) 5'-CCG ATG TTA AAA TCC GTG CAA GCA CTT CAT TT-3' L. brevis Specific 23S rRNA Probes L. brevis Probe 2880 (31mer, 45% G+C) (SEQ ID NO:8) 5'-TGA GGG TTA TTG GTT TCG TTT ACG GGG CTA T-3' L. brevis Probe 2891 (33mer, 48% G+C) (SEQ ID NO:9) 5'-CAG GCT TCC CAA CCT GTT CAA CTA CCA ACA ACT-3' L. brevis Probe 2892 (30mer, 53% G+C) (SEQ ID NO: 10) 5'-CCA CAA TTT GGT GGT ATC CTT AGC CCC GGT-3' L. brevis Probe 2895 (32mer, 53% G+C) (SEQ ID NO:ll) 5'-CAA CCC GGC TGC CAG CAT TTA ACT GGT AAC CT-3' A fourth group of probes is specific to a cluster of Lactobacillus species. A preferred one is given below. Cluster of Lactobacillus sp. 23S rRNA Probe Lactobacillus cluster Probe 2899 (32mer, 47% G+C) (SEQ ID NO: 12) 5'-TCG GTG GAT CAG ATT CTC ACT GAT CTT TCG CT-3' A fifth group of probes can detect both P. damnosus and L. brevis. Preferred ones are given below. P. damnosus and L. brevis 16S rRNA Probes P. damnosus and L. brevis Probe 2904 (30mer, 43% G+C) (SEQ ID NO: 13) 5'-CCA ACA CTT AGC ATT CAT CGT TTA CGG CAT-3' P. damnosus and L. brevis 23S rRNA Probes P. damnosus and L. brevis Probe 2896 (32mer, 44% G+C) (SEQ ID NO: 14) 5'-TTC GCT ACG GCT CCG TTT TTT CAA CTT AAC CT-3' A sixth group of probes hybridizes with the majority of Pediococcus and Lac¬ tobacillus species, and all beer-spoilage organisms. Preferred ones are given below. 16S rRNA Beer-Spoilage Organism Probes Beer-spoilage organism Probe 2873 (28mer, 64% G+C) (SEQ ID NO: 15) 5'-CCC CTG CTT CTG GGC AGG TTA CCC ACG T-3' Beer-spoilage organism Probe 2881 (28mer, 57% G+C) (SEQ ID NO: 16) 5'-TCG CTA CCC ATG CTT TCG AGC CTC AGC T-3' Beer-spoilage organism Probe 2887 (30mer, 63% G+C) (SEQ ID NO: 17) 5'-CGC CGC GGG TCC ATC CAG AAG TGA TAG CCT-3' 23s rRNA Beer-Spoilage Organism Probes Beer-spoilage organism Probe 2875 (32mer, 50% G+C) (SEQ ID NO: 18) 5' CTG AAT TCA GTA ACC CTA GAT GGG CCC CTA GT-3' Beer-spoilage organism Probe 2901 (32mer, 44% G+C) (SEQ ID NO: 19) 5'-TAT CAC TCA CCG TCT GAC TCC CGG ATA TAA AT-3' A seventh group of probes will hybridize to the majority of Pediococcus and Lactobacillus species. Preferred ones are presented below. Majority of Pediococcus and Lactobacillus species 16S rRNA Probes Pediococcus /Lactobacillus Probe 2854 (27mer, 48% G+C) (SEQ ID NO:20) 5'-TAG TTA GCC GTG GCT TTC TGG TTG GAT-3' Pediococcus /Lactobacillus Probe 2879 (28mer, 54% G+C) (SEQ ID NO:21) 5'-CGA TTA CCC TCT CAG GTC GGC TAC GTA T-3' Majority of Pediococcus and Lactobacillus species 23S rRNA Probes Pediococcus /Lactobacillus Probe 2902 (31mer, 58% G+C) (SEQ ID NO:22) 5'-TTC GGG CCT CCA GTG CGT TTT ACC GCA CCT T-3' The probes of the present invention may be used in a "sandwich" assay. As shown in Figure 1, the "sandwich" assay involves use of a pair of probes simultaneously. One probe, designated the "capture" probe 12 is a bifunctional nucleotide made by adding a homopolymeric 3' tail to a probe with preferably high target specificity. The tail will hybridize to the complementary homopolymer 11 on a solid surface 10, such as a glass bead or a filter disc. Hybridization of the capture probe 12 to its target 15, in this case Pediococcus /Lactobacillus rRNA, would complex the target 15 with the solid support 10. The detector probe 13, preferably with some degree of specificity, would be a part of a detection scheme which may use virtually any sort of detection moiety 14, including radioactivity, fluorescence, chemiluminescence, color or other detector moiety. The detector probe may be incorporated as an RNA sequence into an amplifiable Q-beta midivariant as described by Kramer and lizardi, 1989 Nature 339. A sample, such as a swab or liquid aliquot is processed as to liberate the total nucleic acid content. The sample, putatively containing disrupted beer-spoilage or¬ ganisms, is incubated in the presence of a capture probe, detector probe, and magnetic particle beads which have been derivatized with oligo-deoxyThymidine in a chaotropic buffer such as guanidinium isothiocyanate. If target molecules (beer-spoilage microorganisms of the genus Pediococcus or Lactobacillus ) are present, a Bead-Capture Probe-Target-Detector Probe hybridization complex is formed, as in Figure 1. The presence of a magnet near the bottom of the reaction tube will cause the magnetic particle-hybridization complex to adhere to the side of the tube, enabling the removal of the sample matrix, unbound probe, and other constituents not hybridized. Repeated rehydration and denaturation of the Bead- Capture Probe-Target-Detector Probe complex would enable significant background reduction. The final detection may involve spotting the beads on a membrane and assaying by an appropriate method, such as autoradiography, if the detector probe was labelled with a radioisotope. Alternatively, the detector probe may be an amplifiable midivariant probe. The following non-limiting Examples are presented to better illustrate the invention. EXAMPLE 1 Dot-Blot Analysis of Probe Hybridization Behavior , Dot-blot analysis, in accordance with well-known procedures, involves immobiliz¬ ing a nucleic acid or a population of nucleic acids on a filter such as nitrocellulose, nylon or other derivatized membranes which can be readily be obtained commercially. Either DNA or RNA can be so immobilized and subsequently tested for hybridization under a variety of conditions (stringencies) with nucleotide sequences or probes of interest. Under stringent conditions, probes with nucleotide sequences with great com¬ plementarity to the target will exhibit a higher level of hybridization than probes whose sequences have less homology. Probes of the present invention are tested in a dot-blot. One hundred nanograms RNA, is purified by phenol extraction and centrifugation through cesium trifluoroacetate gradients, denatured and spotted on a nylon membrane. Probes are isotopically labelled with the addition of a 32 P-Phosphorous moiety to the 5' end of the ohgonucleotide by the established polynucleotide kinase reaction. Hybridization of the probes is conducted at a temperature of 60oC in the presence of 1.08M NaCl, 60mM sodium phosphate and 6mM ethylenediamine tetraacetic acid (EDTA), pH 7.4. Unhybridized probe is removed by washing at a salt concentration of one-third of the hybridization condition. The filters are exposed to X-ray film and the intensity of the hybridization signals is evaluated after three hours of autoradiographic exposure. The following TABLE 1 is a summary of results. P. damnosus probes targeting 16S rRNA Probe 2858: All P. damnosus strains Probe 2861: All P. damnosus strains; one isolate of Lactobacillus. Probe 2867: All P. damnosus strains L. brevis probes targeting the 16S rRNA Probe 2868: L. brevis specific. This probe misses some isolates identified as L. brevis, but this is thought to be to inaccurate identification of some environmental isolates. Probe 2869: L. brevis specific. Group of P. damnosus and L. brevis probes targeting the 16S rRNA Probe 2904: P. damnosus and L. brevis. Also detects L. buchneή and other related species of Lactobacillus. All beer-spoilage organisms targeting 16S rRNA Probe 2873: Majority of Pediococcus and Lactobacillus strains; all but one spoilage isolate. Probe 2881: Majority of Pediococcus and Lactobacillus strains. Also detects many Gram- positive eubacteria. Probe 2887: Majority of Pediococcus and Lactobacillus strains, all spoilage isolates. Group of Majority of Pediococcus and Lactobacillus species probes, targeting 16S rRNA Probe 2854: Majority of Pediococcus and Lactobacillus strains, also two Bacillus species. Probe 2879: Majority of Pediococcus and Lactobacillus strains. Also detects some Gram- positive bacteria. Group of Majority of Pediococcus beer-spoilage organisms, probes targeting the 23S rRNA Probe 2876: Most Pediococcus strains. Also detects some Lactobacillus isolates. Probe 2877: Most Pediococcus strains. Also detects some Lactobacillus isolates. L. brevis probes targeting the 23S rRNA Probe 2880: L. brevis specific. Misses some isolates identified as L. brevis, but this may be due to inaccurate identification of some environmental isolates. Probe 2891: L. brevis specific. Probe 2892: L. brevis specific. Probe 2895 L. brevis specific. Suhgroup of Lactobacillus genus probes targeting 23S rRNA Probe 2899: Most Lactobacillus species. Possibly some Pediococcus strains. Group of P. damnosus and L. brevis probes targeting 23S rRNA Probe 2896: P. damnosus and L. brevis. Also detects a few other species of Lactobacillus All beer-spoilage organisms targeting 23S rRNA Probe 2875: Majority of Pediococcus and Lactobacillus strains, misses some spoilage isolates. Probe 2901: Majority of Pediococcus and Lactobacillus strains, misses some spoilage isolates. Group of Majority of Pediococcus strains and Lactobacillus species, targeting 23S rRNA Probe 2902: Majority of Pediococcus and Lactobacillus strains. Also some Gram-positive eubacteria. The results of the dot blot assay are presented below as TABLE 2.. In this table, + + + + indicates the strongest signals observed; + + + indicates strong signal observed; + + indicates a somewhat weaker, but definitely positive hybridization signal observed; + indicates a weak signal; +- indicates a very weak, barely detectable signal; - indicates no signal observed. ND indicates that this assay was not performed. If a probe binds strongly (either + + + + or + + +) to at least one target, but exhibits a weak hybridiza- tion (+ or +-) to a second target, the probe is considered to substantially hybridize only with the targets giving the + + + + or + + + results. TABLE2 Pedioeoeeua ΛΛΛ Laetobacillua Dot Bloc Hybridization Results Probe 2ISI 2141 2167 1&&0 Pedloeoceus daanoβua 16S Eubacterial Organise Designation Padiococeua daanotui ~7 . aanoeus PS P.daafiosua P10 P.daanoaua P17 P.daanoaua AICC293SB P.pontoaaceua A_trC33316 .pentoaaceus P1I var. inceraedius Pediococcus ap. P140 Pediococcus ap. PlβO Pediococcus ap. P167 Pediococcus ap. P173 Laetβbaelllus delbruaekii L4 L. ruetlvorani - . L.eaaal L14 L.dβlbrueckii L 7 L.fructivoraπs us L.curvatue L2C L.casei L22 Lactαbacillue ap L137 L-____rtQbaci.ll.UB ap L174 Lactobaelllue ap L176 La.etefeaeillua ap LI77 I__.ecalaa.c-.llus ap L17B Lactabacillua ap L179 LACte.Mtelll.ua sp, L1B5 X_aetobaeilluβ ap, tits -Lactottaeillus ap, L194 poil ge isolate Peβ.ioC490f MD MD Spoilage isolate Pedio53494 MD MD Spoilage isolate PadleC30β55 MD Spoilage isolate PadloC33βar KD ID Spoilage isolate Pe ioM47 NO MD Spoilage isolate 86665 MD MD Spoilage iaolata l__etoC58B*B MD MD Spoilage iaolata LaetoS34S3 MD MD Spoilage iaolate LaetoCSM4λ ID MD Spoilage iaolate LaetoCSlS2 KD ID Spoilage iaolate C490S MD MD Spoilage iaolate LaetoC332S MD ID Spoilage iaolate 10 Laeto aaall MD MD Spoilage iaolata 13 E___cto large D Spoilage iaolata L. brevis CT44.S4 MD Spoilage isolate L. easei CT4C97 ND MD Spoilage iaolate L. brevla CT4698 MD MD Spoilage iβo L. easel CX4S99 MD MD Spoilage isolate - L. brevis CT470Q MD MD Spoilage isolate 3 L. brevis CT4702 MD MD Spoilage iaolata 3 L. brevis OT4703 MD ID Spoilage isolate L. brevis CT4704 MD ID Spoilage isolate delbruoekU CT4705 MD MD Spoilage iaolate B53 L. fructivorans MD MD Spoilage iaolata S53 L. fructlvoraas MD . eidopbilua ASCC435* L.brtfii AXCCS291 L.buC-Uiarl AXCC1130S L.caaal ASCC393 L.easel ASCC74S9 asp. r-uu-nosus L.Λal_6ruβe__Al ΛZ0C11S42 aap. toulgarieua -17- TABLE 2 (continued) Pediococcua and Lactobacillus Dot Blot Hybridization Results Probe 2BSB 2Θ61 2B67 1660 Pediococcus daanosua 16S | Eubacterial Organise Deaignation L.feraentua AICC933B L.ainutus AICC33367 L.plantarua ATCCB014 L.plantarua ΛTCC14917 Leuconoatoc sp. L*ucol92 Leuco.aaxc-nteroides ATCCB293 Aeatobacter aceti AXCC15973 Aectobacter aceti ATCC23746 Acetobaeter aceti AICC23747 λeetobacter aceti ATCC23748 Aceto.kaftββnii AXCC359S9 Aeβto.liqufaciena A_TCC14B35 MD BD ND λccto.pasteurianus ATCC12877 Aceto.pasteurianus ATCC12B79 Aceto.pa.εteuri-u-u.6 AXCC23650 Aeeto.pasteurianus ATCC2375B Aceto.pasteurianua ATCC23764 Acetα.pastaurianus ATCC23765 Aeeto.pasteurianus AICC23766 AcetB.pasteurianus ATCC23767 Aeeto.pasteurianus ATCC3 4 5 Bacillus coagulans AICC7OS0 B.stearothermophilus ATC 129B0 KD HD MD B.βubtilii ATCC21556 Citrobaeter freundii ATCCB090 Enterobacter aerogenes ATCC1304B E.aggloaerans ATCC27155 E. loacae AICC13047 Flavαbactariun ferrugineua ASCC135 ' 24 Cluconαbacter oxydans AI C11S94 C.oxyd_u.c A CC193S7 G.oxydans ATCC23755 G.oxydans ATCC33446 G.oxydans ΛTCC334 7 Ha nia alvei AICC13337 Klebsiclla oxytoea ATCC131B2 Kleb.t___rrige.u_. ATCC 3257 Lactococeus laetis A_TCC1943S asp. lactia Megaspbaera cerevisiae ATCC43236 MD MD ND Megasphaera carav aiaa ATCC 3254 ND KD ND Mieroeoeeua tristiπaβ AICC27S70 Kicrococ ua variana ATCC1530* Qbcεuabecteriua proteus AΪCC12B41 Pectinatus earevisiiptxilus ATCC29359 Peccinatus risingensis ATCC33332 Proteus airabills AS C29906 Serratia urnuini A-TCC13IS0 Staphyloeoccua βpidarmid β AICC149 0 S aph.sapropbytiβus Λ_rcci5305 Zyaoaonas aobilis ΛXCC31B21 ND ND ND Saccharαayces nrniiiu A-TCC1BI24 Sacc aroaycaa ceravisiae ATCC2341 Saccbaroaryees ceravisiae ATCC36902 hiaay Candida albicans AΪCC11006 Huaan/CaSXi Stool SNA Hheat gera RNA 1 8 TABLE 2 (continued) Pediococcus and Lactobacillus Dot Blot Hybridisation Results Probe 2854 2873 2879 2B81 2887 2904 Pediococcus / Lactobacillus 16S Organise Designation Pediococcus damnosus P2 P.daanoBus PS P.daanoaua P10 P.daanoaua P17 P.daanoaua AXCC2935β P. antαsacaus AXCC33316 P.pβntosacβuβ P18 var. interaedius Pediococcus sp. P140 Pedloeoceus sp. P160 Pediococcus sp. P167 Pediococcus sp. P172 Lactobacillus dalbrueclii L4 . tructxvorans L9 L.casci L 4 L.delbrueckil L17 L. Iruccivorans L19 L.eurvatus L20 L.casei L22 Laetobaeillus sp. L137 Lactobacillus ap. LI74 Lactobacillus sp. L17« Lactobacillus sp. LI77 Lactobacillus ap. L178 Lactobacillus sp. L17S Lactobacillus sp. L1B5 Lactobacillus sp. L193 Lactobacillus sp. L194 tt Spoilage isolate 1 PedioC490B ND ND Spoilage isolate 2 PadiθS3454 ND ND Spoilage iaolate 3 PedieC3Q655 ID ND Spoilage isolate 4 PedioC3303P ND ND Spoilage isolate S PedioM67 ND ND Spoilage isolate 7 B66S5 ND ND Spoilage isolate 8 LactoCSlt4B BD ND Spoilage isolate 9 Lacto534S3 ND ND Spoilage isolate 11 L*ctoC5814A ND ND Spoila e isolate 13 L*ctθC5162 ND ND Spoilage isolate 14 C4908 ND ND Spoilage isolate IS LaetoC3325 ND ND Spoilage isolate 10 taβtβ saa l ND ND Spoilage isolate 12 Laeto largo ND ND Spoilage iaolata D L. brevis GT4696 BD ND Spoilage iaolate X L. casei CT44J7 ND ND Spoilage iaolace F L. brevis CΪ4498 ND ND Spoilage isolas* % L. eaaal GTM99 BD ND Spoilage isolate L. brevis GT4700 MD BD Spoilage isolateJ L. brevis CT4702 BD ND Spoilage isolate J L. brevis CT4703 ND ND Spoilage isolate L. brevis CT4704 MD ND Spoilage iaolata L. dalbrueclcii CT4705 ND ND Spoilage isolate 853 L. fruetiwβranβ ND ND Spoilage isolate 853 L. fruecivαrans ND ND L.acidopbiluβ AXCC4356 L. revis ATCCS291 L.buehnari AXCC1130S L.casei JOCC393 L.casei AICC74S9 ssp. rbaanoaus L. albrueclcii AI C11B42 ssp. bulgaricus TABLE 2 (continued) Pediococcus and Laetohacillus Dot Blot Hybridization Results Probe 2854 2B73 2B79 2BS1 2BB7 2904 Padiocoecus / Lactobacillus 165 Organise Designation L.feraentua AICC933B L.ainutus AΪCC332S7 L. lantarua AICCa014 L.p antarua AICC14917 Lcuconoβtoc sp. Leueol92 Leuco.aesenteroidea ATCC8293 Acacobacter aceti AICC15973 Acetobacrer aceei ΛICC23746 Acetobacter aceti A1CC23747 Aeetobacter aceti ATCC237ΛB Aceto.hansβnii AZCC359S9 Aceto.liqufaciens ATCC14135 ND ND ND BD BD MD λeeto.pastau ianua ΛXCC12B77 Aeeto.pasteurianua AXCC12879 Aeato. aatβurianus ASCC23BS0 Aeeto.pasteu ianua AICC237SB Aceto.pasteu anus AXCC237B4 Aeeto.pasteurianus ΛICC23765 Aceto.pa teur anus AICC23766 Acato.paataurlanus A1CC23767 Aceto.pasteurianua ATCC33445 Bacillus ceag lans AZCC70SO -t- B.εtaarot araophilus AICC129B0 ND ND ND ND ND ND B.subtilis ATCC21S56 Citrebactar freundii ATCCB090 Enterebaccer aeregenes AICC13048 ε.aggloaeranβ ATCC271S5 E-cloacae ΛXCC13047 Flavobaetariua farruginetsa ATCC13524 Clucoπobacter ozydaoa ATCC1189* G.oxydans A CC193S7 G.oxydans ATCC237S5 G.oxydans AICC3344B G-oxydanβ ATCC33447 Hafnia alvai XXCC13337 Klebsiella oxytoca ΛXCC131B2 Klab. arrigana AICC332S7 Laetococeua laetis AXCC1943S ssp. laetis Megaspbaara ceravisiae AICC4323f Megasphaera eerevisiae AICC43254 Micrococeus kristiaaa AICC27570 Microcoeeua variana A2CC1S306 Obeauabartariua proteus AΪCC12B41 Peetinatua earevisiipbilus AICC293B Pactinatus frisingansis ATCC33332 Proteus airabilia ASCC2990C Serratia aarcaseeβs ΛXCC13BB0 Staphylocoecua «pi4ara_idia AXCC14990 Stapb.aaprophyl&ua ATCC1S30S 2yaαaonaa s Bi1 iW AXCC31821 ND MD ND ND ND Sacebaroayees eoraviaiaa ATCC1BB24 Saccbaroaycβs ceravisiae ATCC2341 Sacebaroayees eerevisiae AICC3«902 Chios? Candida alblcana AICC1100C Huoan/CaSXi Stool RNA Wheat gera RNA TABLE 2 (continued) Pediococcus and Lactobacillus Dot Blot Hybridization Results Probe 2975 2876 2B77 2896 2901 2902 Padiocoecus 23S Pedlococcua/Lactobacillus ND ND ND ND ND ND ND ND ND ND ND ND ND ND ITO ND ND ND ND ND ND ND HO ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND MD ND ND ND ND ND ND KD ND ND ssp. rbaanβsus L.dalbrueclcii XXCC11842 asp. bulga ieue TABLE 2 (continued) Pediococcus and Lactobacillus Dot Blot Hybridization Results Probe 2B7S 2B76 2877 2896 2901 2902 Pediococcus 23S Pediococcus/LactobacilluE Organise Designation L.fermβntua ATCC933B L.niπutus ATCC33267 L.plan arua ΛTCCB014 L. lanta ua AICC1491 Leueonostoc sp. Leueol92 Leuco.aesanteroides ATCCB293 Acetobaeter aceti ΛΪCC15973 Acetobacter aceti ATCC23746 Acetobaeter aceti AICC23747 Acetobacter aceti ATCC2374B Aceto.hansenii A CC35959 Aceto.ligu aeians ASCC14835 ND ND ND ND ND ND Aeeto.pas eurianus ATCC12B77 Aeeto.pasteu ianus A3CC12B79 Aeeto.pasteu ianus ATCC23650 Aceto.pasteurianus AICC2375B Aceto. asteurianus AICC23764 Aceto. asteurianus AICC23765 Aceto. asteurianus AXCC23766 Aceto. asteurianus AICC23 67 Aeeto.pasteurianus ATCC33445 Bacillus coagulans ATCC70S0 B.stearotberaopbilus ATCC129B0 ND ND ND ND ND ND B.subtilis ΛXCC21556 Citrebacter freundii WCC8090 Enterαbacter aerogenes A_TCC1304β E.aggloaerana ATCC271SS E.cloacae AICC13047 Flavobaeteriua lerrugineua ΛXCC13524 Gluconobaeter oxydans ATCC11B94- G.oxydans ATCC193S7 Q.oxydans ATCC237SS Cozydans AICC33446 G.oxydans ATCC33447 Hafnia alvei ATCC13337 Klebsiella oxytoea AXCC131B2 Xleb.tcrrigcna ATCC33257 Laetocoeeus laetis AXCC1943S ssp. laetis Megaspbaera eeravisiae AICC43236 ND KD ND ND ND ND Hegasphaera eerevisiae ASCC43254 ND ND ND ND ND KD Hierococeus kristiπac AICC37570 Hicrocoeeus variaaa ATCC15306 ObeauB-baeteriua proteus ASCC12B41 Pectinatus cerarisiipbilus AXCC293S9 Pectinatus frisingeπaia ATCC33332 Proteus airabilis AXCC29906 Sarratia aarceseena AICC13M0 Staphylococcus apidenidis ASCC14990 Seaph.sapropbytleuB ',_. AXCC1530S Zyaoaonas aobilis ΛTCC31B21 ND ND ND ND ND ND Sace aroaycas cereviaiae A-TCC1B824 SaeeKaroayeas ceravisiae AΪCC2341 Sacebaroaycas eerevisiae AJCC36902 Chit-ay Candida albicans ATCCllOOβ Hiα-an/CaSXi. Stool UNA Wheat gera RNA TABLE 2 (Continued) Pediococcus and Lactobacillus Dot Blot Hybridization Results Probe 2810 2891 2892 2895 2899 Lactobacillus 23s Organise Pedioeoeeus daanosus P. aanosus P. aanosus P. aanosus P.daanosus P.pentosaeeua P.pentossceus war. in eraedius Pediococcus sp. Padiocoecus ap. Pediococcua ap. Pediococcus sp. Lactobacillus dalbruaekii • .fructivorans .Casei L.delbruecfcii L.fructivorans L.curvaeuβ L.casai Lactobacillus ap. Lactobacillus sp. Lactobacillus sp. Lactobacillus ap. Lactobacillus ap. Lactαbaeillua ap. Lactαbaeillua sp. Lactobacillus ap. Lactobacillus sp. Spoilage isolate 1 Spoilage iaolata 2 Spoilage isolate 3 Spoilage isolate 4 Spoilage iaolate 5 Spoilage isolate 7 Spoilage iaolate 8 Spoilage isolate .9 Spoilage isolate 11 Spoilage iaolate 13 Spoilage iaolate 14 Spoilage isolate IS Spoilage iaolata '10 Spoilage iaolata 12 Spoilage iaolata 13 Spoilage isolate A Spoilage isolate _F Spoilage isolate 8 Spoilage isolate L. brevis CT470 - Spoilage Isolate J L. brevis CT4702 ND Spoilage isolate J L. brevis CT4703 MD Spoilage isolate i L. brevis CTS704 «»ιι KD 11 IT Spoilage isolate ' L. delbruaekii CT470S - MD Spoilage isolate 852 L. έructlvoruis BD Spoilage iaolata 853 L. iructlvorans - ID L.acidaphllus LTCC4356 - L.brevis JOCC8291 t-ii-r ♦*-!-» • w-* L.buchneri . i ASCC1130S - L.easei Λ__CC3t3 - L.casei- • AΪCC7489 . . . sp. L.delbruecfcii f JOCC11B42 ssp. bulgarlcus j TABLE 2 (Continued) Padioeoeeua and Lsetobaeillus Dot Blot Hybridization Results Probe 28&B 2889 2BB0 2891 2892 2B9S 2899 Laetoaaeillus 1CS I Lsetobaeillus 23S Organise Designation L. eraentua AICC933I L.ainucus A2CC31267 L.plantarua AXCCB014 L.plantana Λ3CC14917 Loueonostαe sp. Laucoll3 Leueo.aeseateroides Λ2CCB293 Acetobaeter aceti AICC1Ϊ97J Acetobaeter aeati ΛSCC2374* Acetobaeter aeati JOCC23747 Acetobaeter aeeti J__tCC33748 Aceto. anββnii 1_CCC35959 Aceto.Ugufaciena ATCC14B35 XD ND Aceto.pasteurianus AXCC13877 Aeeto.pasteurianus ATCC13B79 Aeeto.pasteurianus AXCC236S0 Aceto.pasteurianus ASCC237S8 Aeeto.pasteurianus ASCC237B4 Aeeto.pasteurianus JgCC2378B Ace o.pastaurianus AT C23784 Aeeto.pasteurianua JSCC2J787 Aceto. aβtaurlanuβ ASCC33445 Bacillus eoagulans ASCC7080 B.stearotbaraophilus J_3CC12980 ID ID MD MD XD BD B.subtilis BZCC21S84 Citrobaeter fratatdll JOCCB090 Enterabaeter aarogenes JgCC13048 E.aggloaeraa* ATCC371SB C.eloaeae AΪCCU047 Flavobaetariua feiruginaua ASCC13524 Slueonαbaeter exyαans AZCC11B94 C.osydans ATCC19357 C.o_ryc_ana AXCC237SS C.αxydans AZCC3344B C.osydans ΛTCC33447 Hafaia alvai ATCC1J337 Klebsiella oxytoea JLΪCC13182 Kleb.terrlgena ΛSCC33M7 Laetocoecus laetis AXCC1943S asp. laetis Hegasphaara eerevisiae A3CC43234 XD XD XD MD XD Megaapbaars carevlaiaa A-CCC432S4 XD XD XD XD MD BD H erococcus kristlnaa ATCC27570 Mieroeaeevs variaae JBCC1S30* Obesuabecteritaa proteus ASCC12841 Pectinatus cerevlaiiphilus JBCC292S9 Pectinatus frislagoM- A9CC33133 Proteus alrabilis ASCC2990C Sβrratia aareaseeaa ' Λ2CC13BB0 Stapbyloeoceus epiea-raidls ATCC14I 0 Stapb.saprophyticue AΪCC15105 Zyaoaaaas aobills ATCClltJl MD XD KD KD MD MD Saeebaroayees eβrev±aiaβ A2CC18834 Saccbareaycos eereviaiaβ AΪCC2341 Saeebaroayees eerevisiae AXCC36902 Ch__aay Candida albieanβ ATCC11006 Hvjaan/CaSKi Stool Hi Wheat gera BXA Example 2 Dual Probe Hybridization For in-process testing, detection of specific spoilage organisms amongst the wide variety of normal brewery microflora might be most appropriate. For this type of sandwich assay, the following capture and detector probe sets are examples of preferred pairs or sets. P. damnosus 16S rRNA: Probe 2858 + Probe 2861 Probe 2861 + Probe 2867 L. brevis 16S rRNA: Probe 2868 + Probe 2869 P. damnosus &. L. brevis 16S rRNA: Probe 2904 + Probes 2868 + 2861 Group of all spoilage 16S rRNA: Probe 2881 + Probes 2873 + 2887 Group of majority of Pediococcus and Lactobacillus 16S rRNA: Probe 2854 + Probe 2879 L. brevis 23S rRNA Probe 2880 + Probe 2891 Probe 2892 + Probe 2895 P. damnosus & L. brevis 23S rRNA: Probe 2896 + Probes 2880 + 2876 Group of all spoilage 23S rRNA Probe 2875 + Probes 2901 + 2899 Group of majority of Pediococcus and Lactobacillus 23S rRNA: Probe 2902 + Probes 2875 + 2901. Example 3 Brewery and End-Product Detection of Beer-spoilage organisms A sample, such as a swab or liquid aliquot from a bottle, can, keg or other container is processed to yield DNA A probe of this invention is used in conjunction with the antiparallel complement of a second probe of this invention to enzymatically amplify a segment of a target organism gene encoding Lactobacillus rRNA in a polymer- ase chain reaction. Resultant material is then assayed in a sandwich assay. The polymerase chain reaction can, itself be made either highly specific by employing probe/primers described herein, or the reaction may be made more general using probes such as those described in co-pending USSN 359,158 and then identifying the amplifica¬ tion product as a target organism using a sandwich assay. 5 For end-product testing, more generally targeted probes might be ap¬ propriate since most normal brewery microflora should have been removed or been v^i, inactivated. For this particular assay, the following capture detector and detector probes of preferred pairs: 10 P. damnosus 16S rRNA: Probe 2858 + Probe 2861 Probe 2861 + Probe 2867 L. brevis 16S rRNA: Probe 2868 + Probe 2869 P. damnosus & L. brevis 16S rRNA: Probe 2904 + Probes 2868 + 2861 Group of all spoilage 16S rRNA: Probe 2881 + Probes 2873 + 2887 15 Group of majority of Pediococcus and Lactobacillus 16S rRNA: Probe 2854 + Probe 2879 L. brevis 23S rRNA: Probe 2880 + Probe 2891 Probe 2892 + Probe 2895 P. damnosus & L. brevis 23S rRNA: Probe 2896 + Probes 2880 + 2876 20 Group of all spoilage 23S rRNA: Probe 2875 + Probes 2901 + 2899 Group of majority of Pediococcus and Lactobacillus 23S rRNA: Probe 2902 + Probes 2875 + 2901. 25 Example 4 In situ hybridization as a cytological stain The probes of this invention may be used as a cytological staining reagents. A 30 liquid sample is applied to a microscope slide. After fixation and lysis, hybridization of probes is carried out in situ. For example, Probe 2858 is labelled with a florescent label and used to stain the specimen. If P. damnosus is present in the sample, small fluores¬ cent bodies will be visual under a fluorescent microscope. Example 5 Confirmation of Presence of Beer-spoilage organisms following culture Following a standard cultivation step for Pediococcus /Lactobacillus /beer spoilage organisms such as on modified MRS agar plates (Lawrence et al, 1979, J. Instit. for Brewing 85:119) or in liquid culture enrichment, a sample is tested for the presence of Pediococcus/ actobacillus /beer spoilage organisms. One method is by use of the sandwich assay described in Example 2. Pure culture is not necessary. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Nietupski, Raymond M. Stone, Benjamin B. Weisburg, William G. (ii) TITLE OF INVENTION: Nucleic Acid Probes for the Detection of Bacteria of the Genera Pediococcus and Lactobacillus and Methods for the Detection and the Bacterial Agents Causing Spoilage of Beer (iii) NUMBER OF SEQUENCES: 22 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Amoco Corporation (B) STREET: 55 Shuman Blvd., Suite 600 (C) CITY: Naperville (D) STATE: IL (E) COUNTRY: USA (F) ZIP: 60563 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Giesser, Joanne M. (B) REGISTRATION NUMBER: 32,838 (C) REFERENCE/DOCKET NUMBER: 32,442 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (708) 717-2443 (B) TELEFAX: (708) 717-2430 (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TCACAGCCTT GGTGAGCCTT TATCTCAT 28 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CACTGCATGA GCAGTTACTC TCACACACT 29 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGGCTAGCTC CCGAAGGTTA CTCCACCT 28 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: : CCACAGTCTC GGTAATATGT TTAAGCCCCG GT 32 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CGCTCCAACA GTCCTCACGG TCTGCCTTCA T 31 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CAACGTCTGA ACAGTTACTC TCAAACGT 28 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCGATGTTAA AATCCGTGCA AGCACTTCAT TT 32 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGAGGGTTAT TGGTTTCGTT TACGGGGCTA T 31 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CAGGCTTCCC AACCTGTTCA ACTACCAACA ACT 33 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CCACAATTTG GTGGTATCCT TAGCCCCGGT 30 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CAACCCGGCT GCCAGCATTT AACTGGTAAC CT 32 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TCGGTGGATC AGATTCTCAC TGATCTTTCG CT 32 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CCAACACTTA GCATTCATCG TTTACGGCAT 30 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TTCGCTACGG CTCCGTTTTT TCAACTTAAC CT 32 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CCCCTGCTTC TGGGCAGGTT ACCCACGT 28 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TCGCTACCCA TGCTTTCGAG CCTCAGCT 28 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGCCGCGGGT CCATCCAGAA GTGATAGCCT 30 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CTGAATTCAG TAACCCTAGA TGGGCCCCTA GT 32 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TATCACTCAC CGTCTGACTC CCGGATATAA AT 32 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: TAGTTAGCCG TGGCTTTCTG GTTGGAT 27 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGATTACCCT CTCAGGTCGG CTACGTAT 28 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTCGGGCCTC CAGTGCGTTT TACCGCACCT T 31

Claims: What is claimed is: 1. An isolated and purified nucleic acid which hybridizes preferentially with a rRNA or rDNA of a microorganism which causes spoilage of beer. 2. A nucleic acid according to claim 1 wherein the microorganism is selected from the group consisting of the genera Lactobacillus and Pediococcus. 3. A nucleic acid according to claim 1 which is selected from the group of nucleic acids consisting of those which: a) specifically discriminate between P. damnosus and on-Pediococcus species; b) specifically discriminate between the majority of Pediococcus strains causing beer- spoilage and other species; c) Specifically discriminate between L. brevis and non-Lactobacillus species; d) specifically discriminate between a cluster of Lactobacillus species, said cluster consisting of: L. fructivorans, L. casei, L. curvatus, L. brevis, and L. buchneri, and non- cluster species, e) specifically discriminate between the group consisting of P. damnosus and L. brevis and other species; f) specifically discriminate between the majority of Pediococcus and Lactobacillus species causing beer spoilage and other species; g) specifically discriminate between the majority of Pediococcus and Lactobacillus and related species and other species. 4. A nucleic acid according to claim 1 which hybridizes preferentially to 16S rRNA. 5. A nucleic acid according to claim 1 which hybridizes preferentially to 23S rRNA. SUBSTITUTE SHEET (PULE 2β) 6. A nucleic acid according to claim 1 which is complementary to or homologous with at least 90% of a sequence comprising any ten consecutive nucleotides within sequences selected from the group of sequences defined by the group of probes consist¬ ing of: 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891, 2892, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. 7. A nucleic acid probe which hybridizes preferentially with a rRNA or rDNA of a microorganism which causes spoilage of beer. 8. A probe according to claim 7 wherein the microorganism is selected from the group consisting of the genera Lactobacillus and Pediococcus. 9. A probe according to claim 7 which is selected from the group of probes consist¬ ing of those which: a) specifically discriminate between P. damnosus and non-Pediococcus species; b) specifically discriminate between the majority of Pediococcus strains causing beer-spoilage and other species; c) specifically discriminate between L. brevis and non-Lactobacillus species; d) specifically discriminate between a cluster of Lactobacillus species, said cluster consisting of L. fructivorans, L. casei, L. curvatus, L. brevis, and L. buchneή, and non- cluster species; e) specifically discriminate between the group consisting of P. damnosus and L. brevis and other species; f) specifically discriminate between the majority of Pediococcus and Lactobacillus species causing beer spoilage and other species; and g) specifically discriminate between the majority of Pediococcus and Lactobacillus and related species and other species. 10. A probe according to claim 7 which hybridizes preferentially to 16S rRNA 11. A probe according to claim 7 which hybridizes preferentially to 23S rRNA. 12. A probe according to claim 7 which is complementary to or homologous with at least 90% of a sequence comprising any ten consecutive nucleotides within sequences selected from the group of sequences defined by the group of probes consisting of: 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891, 2892, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. 13. A method of detecting the presence of microorganisms which cause the spoilage of beer comprising the steps: contacting a sample suspected of containing a target with at least one nucleic acid which hybridizes preferentially to rRNA or rDNA of a organism selected from the group consisting of: (a) P. damnosus and non-Pediococcus species; (b) the majority of Pediococcus strains causing beer-spoilage but not other species; (c) L. brevis, but not other Lactobacillus species; (d) a cluster of Lactobacillus species consisting of L. fructivorans, L. casei, L. curvatus, L. brevis, and L. buchneri, but not other species; (e) the group of P. damnosus and L. brevis, but not other species; (f) the majority of Pediococcus and Lactobacillus species causing beer spoilage, but not other species; and (g) the majority of Pediococcus and Lactobacillus and related species, but not other species; imposing hybridization conditions on the sample such that the nucleic acid binds preferentially to the target rRNA or rDNA to form nucleic acid complexes; and detecting the complexes as an indication of the presence of the target organisms. 14. A method according to claim 14 wherein the nucleic acid is at least 90% homologous to a sequence comprising any ten consecutive nucleotides selected from the group consisting of sequences defined by probes 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. TE SHEET RULE 2β) 15. A kit which is used for the detection of the presence of microorganisms which cause the spoilage of beer comprising: a) a set of nucleic acids comprising at least two nucleic acids, each nucleic acid comprising 10 to 250 nucleotides and having a different base sequence composition; wherein each nucleic acid is complementary to or homologous with at least 90% of a sequence comprising any ten consecutive nucleotides selected from the group of sequences defined by probes 2858, 2861, 2867, 2876, 2877, 2868, 2869, 2880, 2891, 2892, 2895, 2899, 2904, 2896, 2873, 2881, 2887, 2875, 2901, 2854, 2879, and 2902. 16. A kit according to claim 14 further comprising reagents, and instructions. SUBSTITUTE SHEET (RULE 2β)

Inventors: NIETUPSKI RAYMOND M, STONE BENJAMIN B, WEISBURG WILLIAM G, NIETUPSKI, RAYMOND, M., STONE, BENJAMIN, B., WEISBURG, WILLIAM, G.
Applicants: AMOCO CORP, AMOCO CORPORATION
Application year: 1994
Main classification: C07H21
IPC codes: C07H21, C12N15, C12Q1